Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2025 Apr 1.
Published in final edited form as: Prostaglandins Other Lipid Mediat. 2023 Dec 22;171:106805. doi: 10.1016/j.prostaglandins.2023.106805

12-HETE activates Müller glial cells: the potential role of GPR31 and miR-29

Mohamed Moustafa 1,2,3, Abraham Khalil 4, Noureldien H E Darwish 1,2, Dao-Qi Zhang 1,2,3, Amany Tawfik 1,2,3, Mohamed Al-Shabrawey 1,2,3
PMCID: PMC10939904  NIHMSID: NIHMS1958099  PMID: 38141777

Abstract

Diabetic retinopathy (DR) is a neurovascular complication of diabetes, driven by an intricate network of cellular and molecular mechanisms. This study sought to explore the mechanisms by investigating the role of 12-hydroxyeicosatetraenoic acid (12-HETE), its receptor GPR31, and microRNA (miR-29) in the context of DR, specifically focusing on their impact on Müller glial cells. We found that 12-HETE activates Müller cells (MCs), elevates glutamate production, and induces inflammatory and oxidative responses, all of which are instrumental in DR progression. The expression of GPR31, the receptor for 12-HETE, was prominently found in the retina, especially in MCs and retinal ganglion cells, and was upregulated in diabetes. Interestingly, miR29 showed potential as a protective agent, mitigating the harmful effects of 12-HETE by attenuating inflammation and oxidative stress, and restoring the expression of pigment epithelium-derived factor (PEDF). Our results underline the central role of 12-HETE in DR progression through activation of a neurovascular toxic pathway in MCs and illuminate the protective capabilities of miR-29, highlighting both as promising therapeutic targets for the management of DR.

Keywords: Diabetic Retinopathy; 12/15-lipoxygenase, 12-HETE; GPR31; miR-29; Müller glial; cells

Introduction

Diabetic retinopathy (DR) is a major neurovascular complication of diabetes and a leading cause of vision loss worldwide [1]. The progression of DR is characterized by the breakdown of the blood-retinal barrier, retinal neovascularization, loss of retinal ganglion cells (RGCs), and neuronal dysfunction, eventually leading to vision impairment or blindness [2]. Despite the availability of various treatment options, including laser photocoagulation, intravitreal anti-VEGF injections, and corticosteroids, the prevalence of DR still remains high [3]. Therefore, a better understanding of the underlying molecular mechanisms of DR and the identification of novel therapeutic targets are crucial for the development of more effective interventions.

Müller glial cells (MCs) are critical for maintaining the integrity of retinal neurovascular structure and glutamatergic and redox homeostasis [4,5]. MCs control the glutamate/glutamine metabolic cycle by glutamine synthetase (GS) to decrease extracellular glutamate and prevent glutamate excitotoxicity [6]. Therefore, MC injury has been correlated with neurovascular dysfunction in DR and glaucoma through dysregulation of the glutamate recycling pathway [7, 8, 9, 10]. Prior studies have failed to clarify the relationship between upstream and downstream proteins from glutamate, although the role of oxidative stress and enhanced inflammation in the pathogenesis of DR is well known.

12/15-lipoxygenase (12/15-LO) is an enzyme that catalyzes the oxidation of polyunsaturated fatty acids, leading to the production of bioactive lipid mediators, such as 12- and 15-hydroxyeicosatetraenoic acids (12- and 15-HETEs) [11]. The gene for 12/15-LO in both humans and mice is termed ALOX15, reflecting the close homology (~78%) and largely overlap in their known biological effects in both species. The 12/15-LO isozymes have historically been called 15-LO-1 (ALOX15) in humans due to the preference for 15-HETE generation and only little amounts of 12-HETE (ratio of 9:1). On the other hand, it is called leukocyte 12-LO in mice (encoded by ALOX15 gene), which produces primarily 12-HETE and small amounts of 15-HETE (ratio of 3: 1). Both these enzymes share 73% amino acid similarity, a similar expression pattern a [12,13,14,15,16]. Our previous studies have shown that 12- and 15-HETEs are upregulated in the diabetic retina and contribute to DR progression by promoting inflammation and oxidative stress and disrupting the delicate balance of retinal vascular endothelial growth factor (VEGF) and pigment epithelium derived factor (PEDF) [17, 18, 19]. However, the precise molecular mechanisms and signaling pathways through which 12- or 15-HETE exerts its deleterious effects on the retina remain incompletely understood. Available evidence indicates that G-protein coupled receptor31 (GPR31) and GPR39 are involved in 12-HETE and 15-HETE signaling, respectively, and may be dysregulated in several diseases, including Alzheimer’s disease, cardiovascular diseases, and diabetic complications [20,21,22,23].

The human genome encodes four closely related transcripts of the miR-29 family: miR-29a, b-1, b-2, and c, collectively known as miR-29 [24]. miR-29 has emerged as a potential key player in DR, particularly its expression is altered in the diabetic retina [25,26]. However, the functional relationship between activated 12/15-LO and miR-29 has not been fully explored. Recent studies found decreased expression of miR-29a/b in both streptozotocin (STZ)-induced diabetic rats and mice and in MCs stimulated by high glucose [27, 28]. It is widely understood that the miR-29 family is largely implicated in neuroprotection [29, 30, 31, 32]. Administration of miR-29a prevented retinal neovascularization in oxygen-induced retinopathy and reduced vascular damage in experimental diabetes [30,33]. In addition, we recently reported a significant reduction in levels of retinal miR-29 in a mouse model of hyperhomocysteinemia [33] characterized by significant retinal neurovascular dysfunction [34].

In this study, we aimed to investigate the direct effect of 12-HETE on MCs focusing on its interaction with miR-29. By analyzing the expression and localization of GPR31 and expression of key inflammatory and oxidative stress markers in various experimental groups, we sought to unravel the complex interplay between 12-HETE, GPR31 and miR-29, and their downstream signaling pathways. Considering the established role of 12/15-LO in DR, this knowledge will contribute to a better understanding of the molecular mechanisms underlying DR and may pave the way for the development of novel therapeutic strategies targeting the 12-HETE and miR-29 pathways.

Materials and Methods

Experimental mice

Research involving experimental mice took place at the Eye Research Institute, Oakland University (Rochester, MI, USA), and received approval from the Oakland University Institutional Animal Care and Use Committee (IACUC) under protocol number 2022–1159. Wild-type (Wt) C57BL/6J and Akita mice (Ins2Akita/+, a model for type-1 diabetes mellitus, DM) were obtained from Jackson Laboratories (Bar Harbor, ME). We performed DNA extraction and genotyping according to Jackson Laboratory protocols and using PCR. We housed mice for six months and exposed them to a regular 12-hour light/dark cycle, with free access to water and food at ambient temperature between 22–24°C. We performed intravitreal injection similar to what was previously described [35]. In summary, we applied pupil dilator (tropicamide 1%, Alcon) and topical anesthesia (proparacaine HCl, Alcon, Fort Worth, Texas) eye drops to anesthetized mice. We dissolved 12-HETE (Cayman Chemical, Ann Arbor, MI) in ethanol and considering the vitreous volume of mouse eye is approximately 5–7 μl [36], we injected 0.5 μl of 1 μM 12-HETE or vehicle to obtain a vitreal concentration of 0.1–0.2 μM HETE /mouse eye. We performed intravitreal injection using a 10-μL Hamilton syringe fitted with a 32-gauge microneedle. We inserted needle tip into the vitreous body at a 45° angle through the sclera in the superior hemisphere of the eyeball, guided by a Stemi DV4 Stereo Microscope (Carl Zeiss, Inc., Thornwood, NY). This method of injection prevented damage to eye structures and retinal detachment [37].

Immunostaining of retinal sections

Eyeballs were collected and fixed in 4% PFA for 2 hours then were embedded in the Optimal cutting temperature compound (OCT cryopreservation compound) and 12 μm thick frozen sections were prepared [38]. For immunofluorescence staining retina sections underwent 60-minute blocking and permeabilization step using BlockAid blocking solution (Invitrogen, Waltham, MA, USA, B10710). After washing, slides were incubated overnight at 4°C with primary antibody (glial fibrillary acidic protein (GFAP), GPR31 (Bioss Antibodies, Woburn, MA, USA, BS-13530R), or vimentin (Invitrogen™, Waltham, MA, USA, MA3–745)) diluted in BlockAid blocking solution overnight followed by washing with PBS before adding the secondary antibody for additional one hour incubation. Retinal sections were then covered using Gold Antifade Mounting medium with DNA stain DAPI (Invitrogen™, Waltham, MA, USA, P36931).

Retinal Müller cells

We purchased Immortalized Rat Retinal Müller Cells (rMC1s) from abm (Richmond, BC, CA, T0576). rMC1s were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with, 1% penicillin/streptomycin, and 10% FBS. To investigate the impact of 12-HETE on glutamate and tumor necrosis factor (TNF)-α expression in rMC-1s, 12-HETE (0.1–0.5 μM in serum-free DMEM) was incorporated into the rMC-1 culture for 24–48 hours. Additional group of rMC1 was subjected to treatment with glutamate (1 mM). At the end of the experiment, rMC1-conditioned media were collected and subjected to enzyme-linked immunosorbent assay (ELISA) for TNF-α and Glutamate detection, while cell homogenates were examined using Western blotting to assess PEDF levels (Invitrogen™, Waltham, MA, USA, PA5–115885). Mouse retinal MCs were isolated as described previously [39] and validated using GS immunoreactivity.

Transfection of Cells

We cultured cells according to standard protocols until they reach the desired confluency. We diluted the mirVana™ miR-29a mimic (Invitrogen™, Waltham, MA, USA) to the desired concentration (30 or 100 nM) medium and the transfection reagent (Lipofectamine™ RNAiMAX, Invitrogen™, Waltham, MA, USA) in serum-free medium (Opti-MEM®, Gibco™, Waltham, MA, USA) according to the manufacturer’s instructions. We gently mixed miR-29a mimic and transfection reagent and incubate at room temperature for 15–20 minutes to allow complex formation. wash the cells with serum-free medium, add the transfection complexes dropwise onto the washed cells then incubate the cells at 37°C in a 5% CO2 incubator. We assessed the efficiency of the miRNA mimic transfection using miRNA mimic negative control (Invitrogen™, Waltham, MA, USA).

Immunostaining of Müller cells

The rMC1 or mouse MCs were rinsed with PBS, then fixed with 4% paraformaldehyde for 10 minutes. The cells were then treated with BlockAid blocking solution for 40 minutes at room temperature to prevent non-specific antigen interactions. Primary antibody against GPR31 or GS (Invitrogen™, Waltham, MA, USA, MA5–27749) was diluted using the blocking solution and incubated overnight at 4°C. The cells were then washed three times with PBS before incubation with fluorescent-labelled secondary antibodies (ThermoFisher Scientific, Waltham, MA) for additional one hour at room temperature. After washing of MCs, the coverslips were mounted using the mounting medium Fluoroshield™ with DAPI (Sigma-Aldrich, St. Louis, MO, USA, F6057). Cell nuclei were stained with DAPI for 5 min. The cells were photographed under fluorescent microscopy.

Flow Cytometry Analysis.

For flow cytometry, the rMC1s were gently suspended and incubated with anti-GPR31 at room temperature for 30 min. Finally, the cells were washed once in PBS containing 0.1% human serum albumin (HSA). The cells were then incubated fluorescent-labelled secondary antibodies (FITC-ThermoFisher) for 30 min then washed again before analyzing by flow cytometry (FACS Aria III, BD Biosciences) equipped with an argon and red diode laser, and analysis was performed using Flowjo software (BD Biosciences).

Protein extraction and Western blot analysis

We homogenized cell and retina samples using RIPA buffer (Thermo Scientific™, USA, 89901) followed by centrifuging at 12,000 ×g for 30 minutes at 4°C. We then determined protein concentration using the Pierce™ BCA Protein Assay Kit (Thermo Scientific, USA, 23227) and 50 μg of protein were then boiled in Laemmli buffer, separated by SDS-PAGE on a gradient gel (Bio-Rad), and transferred to a nitrocellulose membrane for antibody incubation. Cell lysates and retinal homogenates were then exposed to primary antibodies against GPR31, GPR39 (Bioss Antibodies, Woburn, MA, USA) or PEDF. Membranes were then stripped and re-probed for β-actin or vinculin to confirm equal loading, and immunoreactivity intensity was measured by densitometry analysis using Image J software (NIH).

Enzyme-linked immunosorbent assay (ELISA)

The levels of Glutamate and TNF-α in the conditioned medium and glutamate in cultured cells were evaluated using ELISA, following the immunoassay kits’ instructions provided by the manufacturer (R & D, Minneapolis, MN, USA). Capsae-3 and PARP-3 levels in retina were measured using Ella Automated Immunoassay System of the Proteomics Core at Georgia Cancer Center, Augusta University.

Assessment of reactive oxygen species generation (ROS)

We added 10 μmol/L of CellROX (Invitrogen™, Waltham, MA, USA) Green Reagent to rMC1s in each well and mixed vigorously to quantify the amount of reactive oxygen species (ROS). The fluorescence intensity of CellROX which reflects the levels of ROS generation was measured using a plate reader.

Total RNA Extraction and q–PCR

Total RNA was extracted from rMC1s using the miRNeasy micro kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA concentrations and quality were determined using Nano Assay (Thermo Scientific™, USA,). RNA was reverse transcribed using the High-Capacity cDNA RT Kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s protocol. Real-time PCR amplification was performed using the TaqMan Universal PCR Master Mix (Applied Biosystems) on AriaMx R-PCR instrument (Agilent Technologies, Palo Alto, CA) and specific Assay-On-Demand TaqMan assays for mir-29b and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data were analyzed using the comparative cycle number determined as threshold (Ct) against expression of GAPDH as normalizer. All samples were loaded in triplicates.

Statistical Analysis

We performed statistical analyses using GraphPad Prism 8. A two-tailed nonparametric t-test or one-way ANOVA followed by the Tukey or the Fishers LSD test was used to assess differences between various experimental groups. Data is presented as means ± SD, with a p-value < 0.05 deemed statistically significant.

Results

12-HETE activates Müller glial cells and upregulates apoptotic markers in retina

Our previous studies demonstrated upregulation of inflammatory mediators NFκB, IL6 and TNF-α in rMC1 by 12-HETE treatment [12]. Consistent with our previous studies, intravitreal administration of 12-HETE (working concentration of 0.1 μM) caused a notable elevation in the expression of MC stress marker GFAP in comparison to vehicle-injected mice 4 days post-injection, implying that 12-HETE activates MCs (Fig 1A). Activation of MCs by 12-HETE was associated with upregulation of retinal apoptotic markers, caspase-3 and PARP-3 (Fig. 1B). Moreover, in vitro exposure of rMC1 to 12-HETE (0.1, 0.2, or 0.5 μM) led to a substantial rise in glutamate production after 24 hours in comparison to the control group, with the most pronounced effect at a concentration of 0.1 μM (Fig 1C).

Fig. 1.

Fig. 1.

Open in a new tab

A) Immunofluorescence of mouse retinal sections stained with glial cell marker GFAP. Intravitreal injection of 12-HETE (0.1μM) induced marked GFAP immunoreactivity compared to vehicle injected contralateral eye. B and C) Multiplex analysis of apoptotic markers caspase-3 and PARP-3 showing significant increase by intravitreal injection of 12-HETE compared to the vehicle (control). D) Treatment of cultured rat Müller cells (rMC1s) with 12-HETE induced significant increase of glutamate production compared to the control. The maximum effect of 12-HETE was noticed at concentration of 0.1 μM compared to 0.25 or 0.5 μM.*P<0.05, ** P=0.004, ****P<0.0001. n=4–6

Determination the expression of a 12-HETE receptor in retinal cells.

To understand the mechanism by which 12-HETE activates MCs and contribute to retinal dysfunction, we investigated the presence and localization of 12-HETE receptor GPR31 in retina. Immunofluorescence staining showed the expression of GPR31 presumably in inner nuclear layer and ganglion cell layer of mouse retina. Colocalization of GPR31 with glial cell marker vimentin showed a remarkable colocalization of GPR31 with vimentin throughout the inner nuclear and ganglion cell layer (Fig 2A). Moreover, colocalization of GPR31 with the ganglion cell marker RBPMS showed a marked colocalization of GPR31 with RBPMS (Fig. 2B). Similarly, immunofluorescence of cultured rMC1s showed expression of GPR31 in the cytoplasmic compartment (Fig 3A). The expression of GPR31 in rMC1 was confirmed using Flow Cytometer which showed high expression of GPR31 in rMC1s (>90%) (Fig 3B). Furthermore, we isolated and characterized mouse MCs (Fig 3C–E) and GPR31 expression was also identified in mouse MC by immunostaining.

Fig. 2.

Fig. 2.

Open in a new tab

Immunolocalization of GPR31 in mouse retinal section. Immunostaining of retinal sections with GPR31 and glial cell marker vimentin showing a marked colocalization in inner nuclear and ganglion cell layers (Arrows in A). Immunolocalization of GPR31 and ganglion cell marker RBPMS also showing colocalization (Arrows in B).

Fig. 3.

Fig. 3.

Open in a new tab

Immunoreactivity of GPR31 in rMC1s showing marked cytosolic localization (A). Expression of GPR31 in rMC1s was confirmed by Flow Cytometer (B). Isolation and culture of mouse Müller cells (brightfield, C). The phenotype of mouse Müller cells is confirmed by glutamine synthase (GS) immunoreactivity (D). GPR31 immunofluorescence in mouse MCs showing marked cytosolic immunoreactivity (E).

Diabetes upregulates the expression of a 12-HETE receptor in mouse retina

Our previous studies have established the role of 12/15-lipoxygenase-derived metabolites in the pathogenesis of diabetic retinopathy. Thus, we tested the effect of diabetes on levels of retinal 12-receptor, GPR31. Western blot examination of retinal homogenates from 6-month diabetic akita mice revealed a significant upregulation of GPR31 (Fig 4). Interestingly, our data also showed upregulation of GPR39, a suggested receptor for the 15-HETE.

Fig. 4.

Fig. 4.

Open in a new tab

Western blot analysis of GPR31 and GPR39 in retinal homogenate of normal and 6-month diabetic (Akita) mice showing significant increases in diabetic group. **P=0.001, ***P=0.0005, n=4.

miR-29 mitigates pro-inflammatory and -oxidative effect of 12-HETE on rMC1s.

We have previously shown that administration of 12-HETE to experimental mice induces neurodegeneration, neovascularization and inflammatory and oxidative effects [12]. Here, we aimed to evaluate a novel approach for reducing the inflammatory and oxidative impact of 12-HETE on glial cells. In vitro exposure of rMC1 to 12-HETE triggered an inflammatory response as shown by significant increase of TNFα production. Effect of 12-HETE on TNFα was comparable to the effect of glutamate (Fig 5 A). Moreover, 12-HETE significantly increased generation of reactive oxygen generation (ROS) in rMC1s (Fig 5B). Effect of 12-HETE on TNFα and ROS was partially mitigated by miR-29a mimic.

Fig. 5.

Fig. 5.

Open in a new tab

ELISA of TNFα in rMC1s condition medium (A). Treatment of rMC1s with glutamate (1 mM) or 12-HETE (0.1 μM) significantly Increased TNFα levels. Effect of glutamate and 12-HETE was reversed by miR-29a mimic. *P<0.05, **P=0.002, n=4–7. Measurement of reactive oxygen species (ROS) in rMC1s using CellRox assay (B) showing significant increases by 12-HETE (0.1 mM) which was also reversed by miR-29a mimic. *P<0.05, n=7

miR-29 preserves PEDF expression in glutamate-treated rMC1

Our previous studies demonstrated suppression of PEDF expression in rMC1s by 12-HETE [12] which also upregulated glutamate production. Here, our data showed that glutamate also induced similar suppressive effect on PEDF levels in rMC1s. Interestingly, this inhibitory effect of glutamate on PEDF expression was significantly abrogated by miR-29a mimic (Fig 6).

Fig. 6.

Fig. 6.

Open in a new tab

Western blot analysis of PEDF expression in rMC1 showing significant reduction by glutamate treatment (1 mM) which was reversed by miR-29a mimic. *P<0.05, n=5–7.

12-HETE attenuates miR29 expression in Müller cells

Recent studies showed decreased expression of miR-29a/b in both streptozotocin (STZ)-induced diabetic rats and mice and in MCs stimulated by high glucose [27, 28]. Here, we examined the effect of 12- and 15-HETE on the expression level of miR29b in rMC1 by RT-PCR. Treatment of rMC1 for 48 hours by 12-HETE showed a significant reduction of miR29b level compared to the control. However, 15-HETE showed a modest reduction in the level of miR29b (Fig. 7).

Figure 7.

Figure 7.

Open in a new tab

RT-PCR of miR29b in rMC1 showing significant reduction by 12-HETE. 15-HETE showing a modest decrease in miR29b expression compared to the effect of 12-HETE. **P=0.002, n=3.

Discussion

Our previous studies provided evidence for the role of 12/15-LO-derived metabolites (12- and 15-HETEs) in DR via promoting inflammatory and oxidative responses [17, 18, 19, 40]. However, there is still a gap in our understanding of the underlying mechanism. The goal of the current study was to characterize the direct effect of 12-HETE on MCs, a key player in mediating neurovascular injury in DR, expression and localization of GPR31 in retina under normal and diabetic conditions and to examine the effect of miR-29a on 12-HETE-induced inflammatory and oxidative responses. Our study demonstrated: 1) Upregulation of glutamate, inflammatory and oxidative responses in MCs by 12-HETE; 2) Expression of 12-HETE receptor GPR31 in retina, mostly in Müller and ganglion cells, 3) diabetes upregulates GPR31, and 4) miR-29a mitigates the pro-inflammatory and -oxidative effects of 12-HETE in MCs.

12/15-LO, a lipid-oxidizing enzyme, produces eicosanoids (bioactive lipids) from ω6 fatty acids (FA) such as arachidonic acid (AA) and ω3 FA such as eicosapentaenoic acids (EPA) and docosahexaenoic acids (DHA). 12/15-LO converts AA to proinflammatory mediators, the 12- and 15-HETEs, while it metabolizes ω3 FA to generate anti-inflammatory and neuroprotective mediators: notably resolvins and neuroprotectins [41,42]. It is highly likely that in diabetes, cardiovascular and neurodegenerative diseases, the ratio between DHA&EPA or their metabolites to AA or its metabolites tilts more towards pro-inflammatory products of AA20. Evidence shows the damaging neurovascular effects of ω6 metabolites versus the protective effects of ω3 metabolites in DR [43,44].

Our previous studied have demonstrated upregulation of retinal 12/15-LO expression and its metabolites 12- and 15-HETEs in both experimental and human diabetes [17, 12]. Moreover, treatment of retinal endothelial cells and MCs with 12-HETE led to activation of inflammatory and oxidative pathways [18]. Since MCs represent the key retinal cells that play crucial role in retinal neurovascular dysfunction in DR, we first tested the direct effect of 12-HETE on MCs both in vivo and in vitro. Intravitreal administration of 12-HETE activated MCs, as marked by elevated expression of GFAP and retinal levels of apoptotic markers capase-3 and PARP-3 underscoring the direct role of this bioactive lipid mediator in MC activation and subsequent disruption of neurovascular homeostasis. This finding aligns with our previous studies, which have implicated 12-HETE in microvascular dysfunction in DR [12–17].

Disruption of glutamate metabolism in MCs and subsequent glutamate excitotoxicity plays essential role in neurodegeneration associated with DR [45, 46]. Our data show that exposure of MCs to 12-HETE leads to a significant increase in glutamate production, a mechanism that could link upregulation of retinal 12/15-LO expression and activity during diabetes to neuronal damage in DR. The positive effect of 12-HETE on TNFα, ROS generation and glutamate production further emphasizes the mechanistic role of 12-HETE in the context of DR.

Accumulated evidence supports GPR31 as a potential receptor for 12-HETE in various tissue that mediates the physiological and pathological effects of 12-HETE [20, 21]. Another pivotal finding in our study is the identification of the GPR31 receptor in retina. Notably, GPR31 is predominantly expressed in MCs and RGCs. This provides a potential mechanistic link between 12-HETE and its downstream effects, highlighting a new therapeutic target to ameliorate 12-HETE-induced retinal damage in DR. Moreover, GPR39 has been suggested as a receptor for 15-HETE [22]. Interestingly, we found that both GPR31 and GPR39 are upregulated in retina of diabetic mice, suggesting that both receptors may contribute to the neurovascular dysfunctional effects of 12- and 15-HETEs during DR.

The interplay between 12-HETE and miR-29 has also been elucidated in this study. miR-29 has been shown to elicit neurotrophic and vasculo-protective effects in various tissues including brain and retinal diseases [29, 30]. Recent studies have also shown that miR-29a is expressed in MCs and modulated under diabetic conditions [27]. Therefore, we sought to examine whether miR-29a attenuates the pro-inflammatory and pro-oxidative effects of 12-HETE in MCs. Treatment of MCs with miR-29a mimic abrogated the inflammatory and oxidative effect of 12-HETE in MCs. Expression of miR-29a/b decreased in both diabetic rats and mice and in MCs stimulated by high glucose [27, 28]. Regardless of different genomic and sequence difference, miR29a and miR29 have similar neurovascular protective function that links them to DR. Our data showed a significant reduction of miR29b in rMC1 by 12-HETE treatment (Fig. 7). Taken together our data and previous studies, miR-29 family has the potential to counteract the detrimental effects of 12-HETE in DR. This could be through its direct interaction with the 12/15-LO-GPR31 or through the regulation of other downstream target genes involved in inflammation and oxidative stress. Moreover, the restoration of PEDF expression by miR-29a mimic under the suppressive influence of glutamate also suggests a protective role of miR-29a in maintaining the neurovascular homeostasis in the diabetic retina. Particularly, PEDF is a known angiostatic and neurotrophic factor in retina [47, 48]. It is released by many of retinal cells including retinal pigment epithelium and MCs [49, 50]. These data are consistent with our previous study that demonstrated suppressive effect for 12-HETE on PEDF expression in MCs, restoration of PEDF levels in retina of oxygen-induced retinopathy model by pharmacological or genetic inhibition of 12/15-LO, and attenuation of neovascularization, inflammatory and oxidative responses to 12-HETE both in vivo and in vitro by PEDF [16]. Taken together our current and previous studies, increased glutamate production by 12-HETE might play a role in the suppressive effect of 12-HETE on PEDF expression in MCs which could be reversed by miR-29.

Overall, the findings presented here underline the importance of 12-HETE in MC activation, the induction of inflammation, and excess generation of glutamate and ROS which are linked to the established role of 12/15-LO in the pathogenesis of DR. the predominant expression of 12-HETE receptor GPR31 in MCs provides a mechanistic insight on how 12-HETE upregulates neurovascular toxic pathways in MCs such as inflammatory, glutamate and oxidative stress. Expression of GPR31 in RGCs also indicates that 12-HETE might elicit direct neuronal injury effect in addition to the neurotoxic effects through MCs. Importantly, the protective effects of miR29 against 12-HETE-induced inflammation and oxidative stress indicate a potential therapeutic strategy for combating the deleterious effects of 12-HETE. However, more research is needed to fully understand the interaction between 12-HETE, GPR31 and miR-29 in DR. Of note, our previous studies demonstrated that retinal endothelial cells could be a major source of 12- and 15-HETEs in DR [17]. This suggest that endothelial-derived HETEs may elicit paracrine effects on MCs and RGCs probably via GPR31/GPR39 to promote neurovascular dysfunction in DR.

Conclusion:

In conclusion, 12-HETE activates MCs, a pivotal step in the progression of retinal damage in DR. It does so by elevating glutamate production, contributing to a neurotoxic environment, and provoking a pro-inflammatory and -oxidative responses, both of which can exacerbate retinal damage. The upregulation of 12- and 15-HETEs receptor, GPR31, and GPR39 respectively in retina of diabetic mice further support the role of 12/15-LO in DR. Our study also reveals the protective role of miR-29, showing that it can counteract the deleterious effects of 12-HETE on retinal MCs, both in terms of inflammation and oxidative stress. Furthermore, the miR-29 mimic’s ability to restore PEDF expression underlines its potential as a novel therapeutic approach in managing DR. Our findings highlight the potential of targeting 12/15-LO and miR-29a signaling as a promising therapeutic strategy in DR. Further research is required to delve deeper into these molecular mechanisms and to design miR-29-based interventions that could improve the clinical management of DR. The onset and progression of DR involve a complex interplay of cellular and molecular mechanisms, many of which are still poorly understood. The results of this study provide valuable insights into the roles of 12/15-LO, miR-29, and their interaction in the pathogenesis of DR, with a particular focus on their effects on MCs (Figure 8). Future studies should consider the systemic effects of manipulating 12-HETE and miR-29 signaling, as both are involved in a broad range of physiological and pathological processes.

Figure 8.

Figure 8.

Open in a new tab

Summary: In diabetes 12-HETE induces glutamatergic, inflammatory, and oxidative responses in Müller cells that may contribute to neurovascular dysfunction in diabetic retinopathy through GPR31. Effect of 12-HETE could be mitigated by the miR-29a. (Solid lines = excitation, a broken line= inhibition)

Highlights.

  • 12-HETE metabolite of the 12/15-lipoxygenase induces glutamatergic, inflammatory, and oxidative responses in Müller cells

  • GPR31, a suggested receptor for 12-HETE is expressed in retina particularly in ganglion and Müller cells.

  • Diabetes upregulates GPR31 in mouse retina.

  • miR29 attenuates the inflammatory and oxidative responses of 12-HETE in Müller cells.

  • 12-HETE/GPR31/miR29 signaling is a potential player in the development of neurovascular dysfunction during diabetic retinopathy through activation of glutamatergic, inflammatory, and oxidative responses in Müller cells.

  • Targeting 12-HETE/GPR31/miR29 has the potential to prevent or at least minimize the neurovascular damage in DR.

Acknowledgment

The work was supported by start-up funds from Oakland University (OU) and Oakland University William Beaumont School of Medicine (OUWB) and NIH/NEI R01 EY030054 to MA. Some experiments were performed using the OUWB Eye Research Center and OU Eye Research Institute Core Facility.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References:

  • [1].Sinclair SH, Schwartz SS. Diabetic Retinopathy-An Underdiagnosed and Undertreated Inflammatory, Neuro-Vascular Complication of Diabetes. Front Endocrinol (Lausanne). 2019;10:843. Published 2019 Dec 13. doi: 10.3389/fendo.2019.00843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [2].Taurone S, De Ponte C, Rotili D, et al. Biochemical Functions and Clinical Characterizations of the Sirtuins in Diabetes-Induced Retinal Pathologies. Int J Mol Sci. 2022;23(7):4048. Published 2022 Apr 6. doi: 10.3390/ijms23074048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [3].Simó R, Hernández C. Novel approaches for treating diabetic retinopathy based on recent pathogenic evidence. Prog Retin Eye Res. 2015;48:160–180. doi: 10.1016/j.preteyeres.2015.04.003. [DOI] [PubMed] [Google Scholar]
  • [4].Bringmann A, Pannicke T, Grosche J, et al. Müller cells in the healthy and diseased retina. Prog Retin Eye Res. 2006;25(4):397–424. doi: 10.1016/j.preteyeres.2006.05.003. [DOI] [PubMed] [Google Scholar]
  • [5].Toft-Kehler AK, Skytt DM, Kolko M. A Perspective on the Müller Cell-Neuron Metabolic Partnership in the Inner Retina. Mol Neurobiol. 2018;55(6):5353–5361. doi: 10.1007/s12035-017-0760-7. [DOI] [PubMed] [Google Scholar]
  • [6].Pow DV, Robinson SR. Glutamate in some retinal neurons is derived solely from glia. Neuroscience. 1994;60(2):355–366. doi: 10.1016/0306-4522(94)90249-6. [DOI] [PubMed] [Google Scholar]
  • [7].Coughlin BA, Feenstra DJ, Mohr S. Müller cells and diabetic retinopathy. Vision Res. 2017;139:93–100. doi: 10.1016/j.visres.2017.03.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [8].Jindal V Neurodegeneration as a primary change and role of neuroprotection in diabetic retinopathy. Mol Neurobiol. 2015;51(3):878–884. doi: 10.1007/s12035-014-8732-7. [DOI] [PubMed] [Google Scholar]
  • [9].Santiago AR, Rosa SC, Santos PF, Cristóvão AJ, Barber AJ, Ambrósio AF. Elevated glucose changes the expression of ionotropic glutamate receptor subunits and impairs calcium homeostasis in retinal neural cells. Invest Ophthalmol Vis Sci. 2006;47(9):4130–4137. doi: 10.1167/iovs.06-0085. [DOI] [PubMed] [Google Scholar]
  • [10].Santos-Carvalho A, Elvas F, Alvaro AR, Ambrósio AF, Cavadas C. Neuropeptide Y receptors activation protects rat retinal neural cells against necrotic and apoptotic cell death induced by glutamate. Cell Death Dis. 2013;4(5):e636. Published 2013 May 16. doi: 10.1038/cddis.2013.160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [11].Kuhn H, Banthiya S, van Leyen K. Mammalian lipoxygenases and their biological relevance. Biochim Biophys Acta. 2015;1851(4):308–330. doi: 10.1016/j.bbalip.2014.10.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [12].Ibrahim AS, Tawfik AM, Hussein KA, et al. Pigment epithelium-derived factor inhibits retinal microvascular dysfunction induced by 12/15-lipoxygenase-derived eicosanoids. Biochim Biophys Acta. 2015;1851(3):290–298. doi: 10.1016/j.bbalip.2014.12.017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [13].Othman A, Ahmad S, Megyerdi S, Mussell R, Choksi K, Maddipati KR, Elmarakby A, Rizk N, Al-Shabrawey M: 12/15- Lipoxygenase-derived lipid metabolites induce retinal endothelial cell barrier dysfunction: contribution of NADPH oxidase. PLoS One 2013, 8:e57254. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [14].van Leyen K, Arai K, Jin G, Kenyon V, Gerstner B, Rosenberg PA, Holman TR, Lo EH: Novel lipoxygenase inhibitors as neuroprotective reagents. J Neurosci Res 2008, 86:904–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [15].Haeggström JZ, Funk CD. Lipoxygenase and leukotriene pathways: biochemistry, biology, and roles in disease. Chem Rev. 2011;111(10):5866–5898. doi: 10.1021/cr200246d. [DOI] [PubMed] [Google Scholar]
  • [16].Adili R, Tourdot BE, Mast K, et al. First Selective 12-LOX Inhibitor, ML355, Impairs Thrombus Formation and Vessel Occlusion In Vivo With Minimal Effects on Hemostasis. Arterioscler Thromb Vasc Biol. 2017;37(10):1828–1839. doi: 10.1161/ATVBAHA.117.309868. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [17].Al-Shabrawey M, Mussell R, Kahook K, et al. Increased expression and activity of 12-lipoxygenase in oxygen-induced ischemic retinopathy and proliferative diabetic retinopathy: implications in retinal neovascularization. Diabetes 2011;60:614–624. Diabetes. 2013;62(3):998. doi: 10.2337/db13-er03. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [18].Elmasry K, Ibrahim AS, Saleh H, et al. Role of endoplasmic reticulum stress in 12/15-lipoxygenase-induced retinal microvascular dysfunction in a mouse model of diabetic retinopathy. Diabetologia. 2018;61(5):1220–1232. doi: 10.1007/s00125-018-4560-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [19].Ibrahim AS, Saleh H, El-Shafey M, et al. Targeting of 12/15-Lipoxygenase in retinal endothelial cells, but not in monocytes/macrophages, attenuates high glucose-induced retinal leukostasis. Biochim Biophys Acta Mol Cell Biol Lipids. 2017;1862(6):636–645. doi: 10.1016/j.bbalip.2017.03.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [20].Guo Y, Zhang W, Giroux C, et al. Identification of the orphan G protein-coupled receptor GPR31 as a receptor for 12-(S)-hydroxyeicosatetraenoic acid. J Biol Chem. 2011;286(39):33832–33840. doi: 10.1074/jbc.M110.216564. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [21].Sharma I, Liao Y, Zheng X, Kanwar YS. Modulation of gentamicin-induced acute kidney injury by myo-inositol oxygenase via the ROS/ALOX-12/12-HETE/GPR31 signaling pathway. JCI Insight. 2022;7(6):e155487. Published 2022 Mar 22. doi: 10.1172/jci.insight.155487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [22].Davis CM, Bah TM, Zhang WH, et al. GPR39 localization in the aging human brain and correlation of expression and polymorphism with vascular cognitive impairment. Alzheimers Dement (N Y). 2021;7(1):e12214. Published 2021 Oct 14. doi: 10.1002/trc2.12214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [23].Alkayed NJ, Cao Z, Qian ZY, et al. Control of coronary vascular resistance by eicosanoids via a novel GPCR [published correction appears in Am J Physiol Cell Physiol. 2022 Jun 1;322(6):C1270]. Am J Physiol Cell Physiol. 2022;322(5):C1011–C1021. doi: 10.1152/ajpcell.00454.2021 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [24].Kriegel AJ, Liu Y, Fang Y, Ding X, Liang M. The miR-29 family: genomics, cell biology, and relevance to renal and cardiovascular injury. Physiol Genomics. 2012;44(4):237–244. doi: 10.1152/physiolgenomics.00141.2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [25].Dantas da Costa E Silva ME, Polina ER, Crispim D, et al. Plasma levels of miR-29b and miR-200b in type 2 diabetic retinopathy. J Cell Mol Med. 2019;23(2):1280–1287. doi: 10.1111/jcmm.14030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [26].Zeng F, Luo G, Lu Y, et al. Long non-coding RNA VIM Antisense RNA 1 (VIM-AS1) sponges microRNA-29 to participate in diabetic retinopathy. Acta Diabetol. 2020;57(9):1111–1116. doi: 10.1007/s00592-020-01536-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [27].Zhang J, Wu L, Chen J, et al. Downregulation of MicroRNA 29a/b exacerbated diabetic retinopathy by impairing the function of Müller cells via Forkhead box protein O4. Diab Vasc Dis Res. 2018;15(3):214–222. doi: 10.1177/1479164118756239. [DOI] [PubMed] [Google Scholar]
  • [28].Zhao BW, Dai HY, Hao LN, Liu YW. MiR-29 regulates retinopathy in diabetic mice via the AMPK signaling pathway. Eur Rev Med Pharmacol Sci. 2019;23(9):3569–3574. doi: 10.26355/eurrev_201905_17778. [DOI] [PubMed] [Google Scholar]
  • [29].Khanna S, Rink C, Ghoorkhanian R, et al. Loss of miR-29b following acute ischemic stroke contributes to neural cell death and infarct size. J Cereb Blood Flow Metab. 2013;33(8):1197–1206. doi: 10.1038/jcbfm.2013.68. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [30].Ouyang YB, Xu L, Lu Y, et al. Astrocyte-enriched miR-29a targets PUMA and reduces neuronal vulnerability to forebrain ischemia. Glia. 2013;61(11):1784–1794. doi: 10.1002/glia.22556. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [31].Ripa R, Dolfi L, Terrigno M, et al. MicroRNA miR-29 controls a compensatory response to limit neuronal iron accumulation during adult life and aging. BMC Biol. 2017;15(1):9. Published 2017 Feb 13. doi: 10.1186/s12915-017-0354-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [32].Zhang J, Zeng Y, Chen J, et al. miR-29a/b cluster suppresses high glucose-induced endothelial-mesenchymal transition in human retinal microvascular endothelial cells by targeting Notch2. Exp Ther Med. 2019;17(4):3108–3116. doi: 10.3892/etm.2019.7323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [33].Elmasry K, Mohamed R, Sharma I, et al. Epigenetic modifications in hyperhomocysteinemia: potential role in diabetic retinopathy and age-related macular degeneration. Oncotarget. 2018;9(16):12562–12590. Published 2018 Jan 29. doi: 10.18632/oncotarget.24333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [34].Ganapathy PS, Moister B, Roon P, et al. Endogenous elevation of homocysteine induces retinal neuron death in the cystathionine-beta-synthase mutant mouse. Invest Ophthalmol Vis Sci. 2009;50(9):4460–4470. doi: 10.1167/iovs.09-3402. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [35].Ibrahim AS, El-Remessy AB, Matragoon S, et al. Retinal microglial activation and inflammation induced by amadori-glycated albumin in a rat model of diabetes. Diabetes. 2011;60(4):1122–1133. doi: 10.2337/db10-1160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [36].Remtulla S, Hallett PE. A schematic eye for the mouse, and comparisons with the rat. Vision Res. 1985;25(1):21–31. doi: 10.1016/0042-6989(85)90076-8. [DOI] [PubMed] [Google Scholar]
  • [37].Leon S, Yin Y, Nguyen J, Irwin N, Benowitz LI. Lens injury stimulates axon regeneration in the mature rat optic nerve. J Neurosci. 2000;20(12):4615–4626. doi: 10.1523/JNEUROSCI.20-12-04615.2000 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [38].You Y, Gupta VK, Li JC, Al-Adawy N, Klistorner A, Graham SL. FTY720 protects retinal ganglion cells in experimental glaucoma. Invest Ophthalmol Vis Sci. 2014;55(5):3060–3066. Published 2014 Apr 17. doi: 10.1167/iovs.13-13262. [DOI] [PubMed] [Google Scholar]
  • [39].Liu X, Tang L, Liu Y. Mouse Müller Cell Isolation and Culture. Bio Protoc. 2017;7(15):e2429. doi: 10.21769/BioProtoc.2429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [40].Othman A, Ahmad S, Megyerdi S, et al. 12/15-Lipoxygenase-derived lipid metabolites induce retinal endothelial cell barrier dysfunction: contribution of NADPH oxidase. PLoS One. 2013;8(2):e57254. doi: 10.1371/journal.pone.0057254 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [41].Lazic M, Inzaugarat ME, Povero D, Zhao IC, Chen M, Nalbandian M, Miller YI, Chernavsky AC, Feldstein AE, Sears DD: Reduced dietary omega-6 to omega-3 fatty acid ratio and 12/15-lipoxygenase deficiency are protective against chronic high fat diet-induced steatohepatitis. PLoS One 2014, 9:e107658. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [42].Elmarakby AA, Ibrahim AS, Katary MA, Elsherbiny NM, El-Shafey M, Abd-Elrazik AM, Abdelsayed RA, Maddipati KR, Al-Shabrawey M: A dual role of 12/15-lipoxygenase in LPS-induced acute renal inflammation and injury. Biochim Biophys Acta Mol Cell Biol Lipids 2019, 1864:1669–80. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [43].Chen W, Jump DB, Grant MB, Esselman WJ, Busik JV: Dyslipidemia, but not hyperglycemia, induces inflammatory adhesion molecules in human retinal vascular endothelial cells. Invest Ophthalmol Vis Sci 2003, 44:5016–22. [DOI] [PubMed] [Google Scholar]
  • [44].Sala-Vila A, Diaz-Lopez A, Valls-Pedret C, Cofan M, Garcia-Layana A, Lamuela-Raventos RM, Castaner O, Zanon- Moreno V, Martinez-Gonzalez MA, Toledo E, Basora J, Salas-Salvado J, Corella D, Gomez-Gracia E, Fiol M, Estruch R, Lapetra J, Fito M, Aros F, Serra-Majem L, Pinto X, Ros E, Prevencion con Dieta Mediterranea I: Dietary Marine omega-3 Fatty Acids and Incident Sight-Threatening Retinopathy in Middle-Aged and Older Individuals With Type 2 Diabetes: Prospective Investigation From the PREDIMED Trial. JAMA Ophthalmol 2016, 134:1142–9. [DOI] [PubMed] [Google Scholar]
  • [45].Jindal V Neurodegeneration as a primary change and role of neuroprotection in diabetic retinopathy. Mol Neurobiol. 2015;51(3):878–884. doi: 10.1007/s12035-014-8732-7. [DOI] [PubMed] [Google Scholar]
  • [46].Santiago AR, Rosa SC, Santos PF, Cristóvão AJ, Barber AJ, Ambrósio AF. Elevated glucose changes the expression of ionotropic glutamate receptor subunits and impairs calcium homeostasis in retinal neural cells. Invest Ophthalmol Vis Sci. 2006;47(9):4130–4137. doi: 10.1167/iovs.06-0085. [DOI] [PubMed] [Google Scholar]
  • [47].Yamagishi S, Matsui T, Nakamura K, Takeuchi M, Imaizumi T. Pigment epithelium-derived factor (PEDF) prevents diabetes- or advanced glycation end products (AGE)-elicited retinal leukostasis. Microvasc Res. 2006;72(1–2):86–90. doi: 10.1016/j.mvr.2006.04.002 [DOI] [PubMed] [Google Scholar]
  • [48].Mori K, Duh E, Gehlbach P, et al. Pigment epithelium-derived factor inhibits retinal and choroidal neovascularization. J Cell Physiol. 2001;188(2):253–263. doi: 10.1002/jcp.1114. [DOI] [PubMed] [Google Scholar]
  • [49].King GL, Suzuma K. Pigment-epithelium-derived factor--a key coordinator of retinal neuronal and vascular functions. N Engl J Med. 2000;342(5):349–351. doi: 10.1056/NEJM200002033420511. [DOI] [PubMed] [Google Scholar]
  • [50].Ogata N, Wada M, Otsuji T, Jo N, Tombran-Tink J, Matsumura M. Expression of pigment epithelium-derived factor in normal adult rat eye and experimental choroidal neovascularization. Invest Ophthalmol Vis Sci. 2002;43(4):1168–1175. [PubMed] [Google Scholar]

RESOURCES